Smooth Muscle Cell Proangiogenic Phenotype Induced by Cyclopentenyl Cytosine Promotes Endothelial Cell Proliferation and Migration*

Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) are in close contact with blood vessels. SMC phenotypes can be altered during pathological vascular remodeling. However, how SMC phenotypes affect EC properties remains largely unknown. In this study, we found that PDGF-BB-induced synthetic SMCs suppressed EC proliferation and migration while exhibiting increased expression of anti-angiogenic factors, such as endostatin, and decreased pro-angiogenic factors, including CXC motif ligand 1 (CXCL1). Cyclopentenyl cytosine (CPEC), a CTP synthase inhibitor that has been reported previously to inhibit SMC proliferation and injury-induced neointima formation, induced SMC redifferentiation. Interestingly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration because of an increase in CXCL1 along with decreased endostatin production in SMCs. Addition of recombinant endostatin protein or blockade of CXCL1 with a neutralizing antibody suppressed the EC proliferation and migration induced by CPEC-conditioned SMC medium. Mechanistically, CPEC functions as a cytosine derivate to stimulate adenosine receptors A1 and A2a, which further activate downstream cAMP and Akt signaling, leading to the phosphorylation of cAMP response element binding protein and, consequently, SMC redifferentiation. These data provided proof of a novel concept that synthetic SMC exhibits an anti-angiogenic SMC phenotype, whereas contractile SMC shows a pro-angiogenic phenotype. CPEC appears to be a potent stimulator for switching the anti-angiogenic SMC phenotype to the pro-angiogenic phenotype, which may be essential for CPEC to accelerate re-endothelialization for vascular repair during injury-induced vascular wall remodeling.


PDGF-BB-induced Synthetic SMC Suppressed EC Proliferation and Migration-PDGF-BB is a potent and known SMC mitogen that induces SMC proliferation and migration
These data indicate that synthetic SMC exhibits an antiangiogenic phenotype.
CPEC Induced Synthetic SMC Redifferentiation into a Contractile SMC Phenotype-Our previous studies showed that CPEC inhibits SMC proliferation (29). Because synthetic SMC displays an anti-angiogenic phenotype, and CPEC promotes re-endothelialization without affecting EC proliferation, we sought to determine whether CPEC alters the SMC phenotype. As shown in Fig. 2A, CPEC induced SMC marker gene expression in both vehicle and PDGF-BB-treated synthetic SMCs. In fact, CPEC induced the expression of SMC contractile proteins such as SMMHC and SM22␣ in PDGF-BB-treated SMCs in a dose- (Fig. 2, B and C) and time-dependent manner (Fig. 2, D and E), suggesting that CPEC induces the redifferentiation of synthetic SMC to a contractile SMC phenotype. Because contractile SMCs have a spindle-shaped morphology, we tested whether CPEC treatment also alters SMC morphology. As shown in Fig. 2F, CPEC induced SMCs to assume an elongated and spindle-shaped morphology resembling the SMC phenotype observed in the media layer of the artery. To further verify  the CPEC-induced SMC differentiation, we tested whether CPEC induces SMC progenitor cells to express SMMHC. As shown in Fig. 2, G and H, CPEC stimulated a dose-dependent expression of SMMHC in human embryonic stem cell-derived mesenchymal stem cells (hMSC). These data indicate that CPEC can induce the contractile phenotype from either synthetic SMC or SMC progenitors.
CPEC-conditioned SMC Culture Medium Promoted EC Proliferation and Migration-Because synthetic SMC exhibited an anti-angiogenic effect ( Fig. 1, C-F), we sought to determine whether CPEC-induced contractile SMCs have a pro-angiogenic property. Thus, we tested whether CPEC-treated SMCs express pro-or anti-angiogenic factors. As shown in Fig. 3, A and B, although PDGF-BB blocked expression of the pro-angiogenic factors CXCL1, CYR61, and G-CSF and induced the antiangiogenic factors EST and TSP1, CPEC reversed the effect of PDGF-BB, as shown by stimulating expression of the pro-angiogenic factors while inhibiting expression of the anti-angiogenic factors. Importantly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration (Fig. 3, C-E). These results suggest that CPEC-induced contractile SMCs have a pro-angiogenic property.
CPEC Induced the Pro-angiogenic Effect of SMCs via EST and CXCL1-Because CXCL1 and EST exhibited the most dramatic changes in SMCs (Figs. 1C and 3, A and B), we tested whether CPEC induces the paracrine angiogenic effects of SMCs via EST or CXCL1. PDGF-BB induced EST and down-regulated CXCL1 protein expression in synthetic SMCs (Fig. 4, A and B), consistent with their mRNA expression (Figs. 1C and 3, A and B). CPEC, however, reversed the effect of PDGF-BB, as shown by the inhibition of EST and the increase in CXCL1 protein expression in PDGF-BB-treated SMCs (Fig. 4, C and D). CPEC also regulated EST and CXCL1 protein expression in a time-dependent manner (Fig. 4, E and F). To determine whether CPEC affects EST and CXCL1 secretion in addition to their expres-sion, we detected the EST and CXCL1 protein levels in SMC culture medium via ELISA. As shown in Fig. 4, G and H, CPEC suppressed EST and increased CXCL1 secretion into SMC culture medium with PDGF-BB treatment.
To test whether CXCL1 and EST play roles in the pro-angiogenic effect of CPEC-induced SMCs on EC proliferation and migration, we added recombinant EST or CXCL1-neutralizing antibody (to block CXCL1 function) in CPEC-conditioned SMC culture medium. As shown in Fig. 4, I and J, recombinant EST ( Fig. 4I) or CXCL1-neutralizing antibody (Fig. 4J) effectively suppressed the EC proliferation and migration promoted by the paracrine effects of contractile SMCs that was induced by CPEC, suggesting that CXCL1 and EST mediated the proangiogenic effect of the CPEC-induced SMC phenotype.
CPEC Promoted SMC Redifferentiation through Adenosine Receptor (ADOR) Activation-CPEC is an inhibitor of CTPS. However, blockade of CTPS1 expression by its shRNA did not increase the expression of the contractile SMC marker SM22␣ (Fig. 5, A and B), suggesting that CPEC induced SMC redifferentiation through a CTPS-independent mechanism. In addition to inhibiting CTPS, CPEC can deplete the intercellular CTP pool. Thus, we tested whether CPEC induces SMC redifferentiation by altering the CTP pool. Cytidine feeding has been shown to increase the intercellular CTP concentration (42), so we added cytidine to CPEC-treated SMCs to counter the CPEC effect. Surprisingly, combination treatment of CPEC and cytidine did not attenuate the CPEC effect; rather, it led to higher SM22␣ expression (Fig. 5, C and D). This increased effect as a result of the addition of cytidine prompted us to hypothesize that CPEC may act as a cytosine derivate to stimulate SMC redifferentiation. In support of this hypothesis, other endogenous cytosine derivatives such as cytosine, cytidine, and CMP, exhibited similar effects in inducing SM22␣ expression in SMCs (Fig. 5, E and F). Previous studies have shown that adenosine receptors can function as receptors for nucleotide mimics (43). CPEC upregulated the expression of ADORA1 and ADORA2a in both control and PDGF-BB-treated SMCs (Fig. 6A). CPEC also enhanced ADORA2a protein expression (Fig. 6, B and C). Moreover, the ADORA downstream signal was also activated by CPEC treatment, as evidenced by the increased intercellular cAMP level (Fig. 6D) and the phosphorylation of cAMP response element-binding protein (CREB) (Fig. 6, E and F). Importantly, both the ADORA1 inhibitor CPDX and the ADORA2a inhibitor KW-6002 suppressed CPEC-induced SMC redifferentiation (Fig. 6, G and H), indicating that CPEC regulates SMC redifferentiation through ADORA signaling. Notably, KW-6002 displayed a more potent effect than CPDX (Fig. 6, G and H), suggesting that ADORA2a may share more responsibility than ADORA1 in mediating CPEC function.
CPEC Triggered SMC Redifferentiation through ADOR Downstream Akt Signaling-It is well established that Smad3 activation is critical for contractile protein expression and SMC differentiation (44). However, CPEC did not enhance Smad3 expression or phosphorylation (Fig. 7, A and B), suggesting that CPEC induces SMC redifferentiation through a Smad-independent mechanism. Indeed, CPEC induced Akt phosphorylation in a dose-dependent manner in both control and PDGF-BBtreated SMCs (Fig. 7, A and B). CPDX or KW-6002 suppressed CPEC-induced Akt phosphorylation (Fig. 7, C and D), suggesting that CPEC activates Akt signaling via ADORA1 and ADORA2a. Because Akt signaling is involved in CREB activation (45), which further regulates SMC differentiation (46), we tested whether CPEC induced SMC redifferentiation through the ADORA-Akt-CREB axis. Thus, we tested whether PI3/Akt signaling mediated CPEC-induced CREB phosphorylation. As shown in Fig. 7, E and F, the blockade of PI3K/Akt signaling by itsinhibitorLY-294002attenuatedCPEC-enhancedCREBphosphorylation as well as SMC marker SM22␣ expression (Fig. 7, G and H), indicating that CPEC induced SMC redifferentiation through the ADORA-Akt-CREB axis.
CPEC Induced Neointimal SMC Redifferentiation, Inhibited EST and Enhanced CXCL1 Expression, and Promoted Re-endothelialization in Vivo-CPEC did not promote EC proliferation and migration in vitro (29), but CPEC promoted EC proliferation/migration via the paracrine effect of CPEC-induced con- tractile SMCs. To test whether CPEC-induced contractile SMCs produce angiogenic factors under pathological conditions, we used rat carotid artery balloon injury model to mimic vascular injury in vivo and use an osmotic pump to infuse saline or CPEC into rats undergoing the artery injury. As shown in Fig.  8, A and B, infusion of CPEC significantly attenuated neointima formation and promoted redifferentiation of the neointimal SMCs, as indicated by expression of the SMC marker SM22␣ compared with the saline-treated artery. Importantly, neointimal SMCs in CPEC-treated arteries showed reduced expression of the anti-angiogenic factor EST and a significant increase in pro-angiogenic CXCL1 (Fig. 8, C-F). Consequently, CPEC treatment promoted re-endothelialization, as demonstrated by CD31 staining (Fig. 8, G and H), consistent with our previous finding (29). These data suggest that CPEC induces a contractile/pro-angiogenic phenotype in neointimal SMCs, which promotes EC proliferation/migration, resulting in accelerated re-endothelialization.

Discussion
EC proliferation and migration are key events during vascular repair following injury. The mechanisms underlying re-endothelialization are thought to be attributable primarily to the intrinsic factors or signaling of ECs. This study indicates that SMC phenotypes play critical roles in EC properties. Proliferative or synthetic SMCs inhibit EC proliferation and migration, whereas CPEC-induced contractile SMCs stimulate EC proliferation/migration, which is due to the production of pro-angiogenic factors and the blockade of anti-angiogenic factors within the redifferentiated SMCs. Therefore, contractile SMCs exhibit a pro-angiogenic phenotype, whereas synthetic SMCs display an anti-angiogenic phenotype. This concept is also supported by a previous study showing that serum-treated SMCs suppress EC replication, although the mechanism has not been determined (47). It is likely that serum-treated SMCs act similarly as PDGF-BB-treated SMCs, i.e. producing anti-angiogenic factors to block EC proliferation.
The pro-angiogenic function of CPEC-induced SMCs is likely to be a unique property of contractile SMCs converted from neointima/proliferative SMCs during vascular repair following pathological remodeling. Thus, it may not be relevant to physiological angiogenesis because mature SMCs inhibits excessive EC proliferation and migration during vascular development (27). Accordingly, CPEC-induced SMCs may not be identical to mature SMCs in the blood vessel that is formed during the vascular development, although CPEC can also induce SMC differentiation from mesenchymal progenitors.
Interestingly, although CPEC is an inhibitor of CTPS, it does not cause SMC phenotypic alteration by its function in CTPS activity or depletion of the CTP pool. Instead, CPEC plays a new role by acting as a cytosine derivate to induce SMC redifferentiation. Importantly, other cytosine derivatives can also induce SMC redifferentiation, although their effects are much less compared with CPEC (Fig. 5E). Combined treatment of CPEC and cytidine significantly increases SMC marker gene expression, suggesting that CPEC and other cytosine derivatives have a synergistic effect in inducing SMC redifferentiation. Collectively, it appears that CPEC promotes vascular repair through two independent mechanisms, i.e. blocking SMC proliferation via inhibition of CTPS activity and prompting a contractile/ pro-angiogenic SMC phenotype by acting as a cytosine derivate. Both functions appear to be critical for promoting reendothelialization and vascular repair.
CPEC induces SMC redifferentiation via the ADOR-PI3K/ Akt-CREB axis. ADOR is a class of G protein-coupled purinergic receptors using free nucleotides (mainly adenosine and uridine) as ligands (48). SMCs express ADORs. Functionally, these receptors are involved in cell contraction and ion channel activities related to cAMP signaling (49 -51). Our results demonstrate for the first time that ADORA1 and ADORA2a may also serve as receptors for CPEC, which activates both cAMP and Akt; cAMP and Akt further promote CREB phosphorylation, leading to SMC redifferentiation. The results showing that PI3K/Akt inhibitor blocks CPEC-induced SMC redifferentiation are consistent with previous findings that the PI3K/Akt pathway is essential for maintaining the differentiated SMC phenotype (52) and that cAMP-mediated Akt signaling is necessary for SMC contraction (49 -51). Therefore, PI3K/ Akt signaling may regulate the SMC phenotype via both CREB-dependent and CREB-independent mechanisms.
In summary, we have demonstrated that PDGF-BB-induced synthetic SMC exhibits an anti-angiogenic phenotype inhibiting EC proliferation/migration, whereas CPEC-induced contractile SMCs shows a pro-angiogenic phenotype promoting  DECEMBER 23, 2016 • VOLUME 291 • NUMBER 52

Experimental Procedures
Reagents and Cell Culture-Rat aortic SMCs and ECs were cultured by the enzyme digestion method from rat thoracic aortae as described previously (53,54). SMCs were maintained at 37°C in a humidified 5% CO 2 incubator in DMEM containing 10% FBS, 4.5 g/liter glucose, 4.5 g/liter sodium pyruvate, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml strep-tomycin. Primary ECs were maintained at 37°C in a humidified 5% CO 2 incubator in DMEM containing 20% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 10 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES, 100 g/ml heparin, 100 g/ml endothelial cell growth supplement. Cells within passage 3 were used for all experiments in this study. The SMC and EC phenotypes of the cultured cells were confirmed by expression of smooth muscle ␣-actin and CD31, respectively. hMSCs were obtained from ArunA Biomedical (Athens, GA). hMSCs were cultured in ␣-minimal essential medium  (Cellgro, Fisher Scientific, Pittsburgh, PA) with 10% mesenchymal stem cell-qualified fetal bovine serum (Hyclone) and 2 mM L-glutamine (Hyclone).
SMCs were cultured in complete medium with or without treatment of CPEC and/or PDGF-BB. 48 h after the treatment, 3 ϫ 10 6 cells were reseeded in 6-cm culture dishes. After the SMC monolayer was formed, cells were washed with warm PBS three times and incubated with serum-free DMEM for 12 h, followed by collection of conditioned medium that was filtered through a 0.22-m filter, and stored at Ϫ80°C.
Animals-Male Sprague-Dawley rats weighing 450 -500 g were purchased from Harlan. All animals were housed under conventional conditions in the animal care facilities and received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. Animal surgical procedures were approved by the Institutional Animal Care and Use Committee of the University of Georgia.
Rat Carotid Artery Injury Model and Immunohistochemistry Staining-Rat carotid artery balloon injury was performed using a 2F Fogarty arterial embolectomy balloon catheter (Bax-ter Edwards Healthcare) as described previously (53). 14 days later, the balloon-injured arteries were perfused with saline, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. For immunohistochemistry staining, sections were rehydrated, permeabilized with 0.01% Triton X-100 in PBS, blocked with 10% goat serum, and incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibody. The sections were counterstained with hematoxylin.
Quantitative RT-PCR (qPCR)-Total RNA was extracted from primary cultured SMCs using TRIzol reagent (Invitrogen) and reverse-transcribed to cDNA using the iScript TM cDNA synthesis kit (Bio-Rad). qPCR was performed on a Stratagene Mx3005 qPCR thermocycler (Agilent Technologies, La Jolla, CA) as described previously (55). Primer sequences for qPCR are listed in supplemental Table S1. Cyclophilin was used as an internal control. The primer efficiency was verified by the dissociation curve in qPCR reactions. The -fold change for each target gene was calculated as 2 Ϫ⌬⌬Ct .
Western Blotting-Western blotting was performed as described previously (55).
EC Proliferation Assay-ECs were seeded in 24-well plates at 1 ϫ 10 4 /well in EC complete medium. After 24 h, the medium was changed to DMEM containing 20% SMC-conditioned medium, 2% FBS, and 2 mM L-glutamine and cultured for another 4 days. Fresh medium was changed every other day, and cell numbers were counted every day.
Transwell Assay-The transwell assay was carried out according to the instructions of the manufacturer (Corning Inc., Corning, NY). Primary ECs were seeded onto the transwell upper chamber inserts (1 ϫ 10 5 /insert). The inserts were then put back into the receiver plate filled with 80% endothelial culture medium plus 20% SMC-conditioned medium and incubated at 37°C in a humidified 5% CO 2 incubator for 12 h. After incubation, the transwell inserts were washed with PBS three times and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by washing with PBS. Cells were stained FIGURE 8. CPEC promoted re-endothelialization through inducing neointima SMC redifferentiation in vivo. Rat left carotid arteries were injured with a 2F Fogarty arterial embolectomy balloon catheter. 1 mg/kg/day CPEC was infused into the rat through an osmotic minipump that was implanted on the day of artery injury. 14 days later, the arteries were removed, and immunohistochemistry staining with the indicated antibodies was performed. A and B, CPEC significantly increased contractile SMC marker SM22␣ expression in neointimal SMCs. C and D, CPEC suppressed expression of the anti-angiogenic factor EST in neointimal SMCs. E and F, CPEC induced expression of pro-angiogenic factor CXCL1 expression in neointimal SMCs. G and H, CPEC accelerated re-endothelialization. The endothelium was stained by CD31. The expression of SM22␣, EST, and CXCL1 was quantified by measuring mean intensity per area in 10 different fields and is shown as -fold changes. The re-endothelization was quantified by averaging the CD31-positive cells in 10 different fields and is shown as -fold changes. *, p Ͻ 0.05; **, p Ͻ 0.01; n ϭ 10.
with Wright-Giemsa for acquiring images or DAPI for cell counting. Images of migrating cells were captured using a dissection microscope (Olympus).
ELISA-CXCL1 and EST levels in SMC-conditioned medium were measured with commercial rat CXCL1 (MBS824537) and Endostatin ELISA kits (MBS730385) from MyBioSource Inc., respectively. SMC-conditioned medium was collected, filtered through a 0.22-m filter, and stored at Ϫ80°C for later use. Purified rat CXCL1 and EST were serially diluted and served as standards. Intercellular cAMP levels were measured with a commercial cAMP ELISA kit (EIA-CAMP-1) from RayBiotech, Inc. ELISA assays were performed according to the protocol of the manufacturer.
Statistical Analysis-Each experiment was repeated at least three times. All values are presented as mean Ϯ S.E. Comparisons of parameters among groups were made by one-way analysis of variance, and comparisons of different parameters between each group were made by a post hoc analysis using a Bonferroni test. p Ͻ 0.05 was considered statistically significant.
Author Contributions-R. T. and S. Y. C. conceived and coordinated the study. R. T. designed and performed the experiments shown in Figs. 1-8. G. Z. provided technical assistance and contributed to the experiments shown in Figs. 2 and 4 -6. R. T. and S. Y. C. analyzed the results and wrote the paper. All authors analyzed the results and approved the final version of the manuscript.